Agonist binding to GPCRs causes rapid phosphorylation of the activated receptors by GPCR kinases. This process promotes the recruitment of members of the arrestin protein family (beta-arrestin-1 and -2) to the activated receptors, disrupting receptor/G protein coupling and promoting GPCR internalization by targeting the receptors to clathrin-coated pits. However, during the past 10-15 years, many studies have demonstrated that beta-arrestins can also act as signaling molecules in their own right. This observation is not only of theoretical interest but also of potential clinical relevance. Despite recent advances in understanding the molecular mechanisms of beta-arrestin function, the potential interplay of heterotrimeric G proteins with beta-arrestin-mediated processes remains to be investigated. Inactivation and/or deletion of heterotrimeric G proteins prevents beta-arrestin-dependent signaling In a recent collaborative study, we asked the question whether beta-arrestin signaling is independent of the presence of functional heterotrimeric G proteins. To address this issue, we used CRISPR/Cas9 technology to delete G proteins of the Gs, Gq and G12 families in HEK293 cells. In addition, we inactivated G proteins of the Gi family by treating HEK293 cells with pertussis toxin. Moreover, we generated HEK293 cells deficient in both beta-arrestin-1 and -2. We then used these engineered cells to systematically dissect G protein- from beta-arrestin-driven signaling outcomes for a wide range of GPCRs. For these studies, we used various biochemical and biophysical techniques including label-free whole cell biosensing. Particular emphasis was placed on mechanisms underlying signaling via the extracellular signal regulated kinase 1/2 (ERK1/2) cascade, one of the most prominent examples for G protein-independent, beta-arrestin-dependent signaling. Interestingly, all ligand-activated GPCRs were able to recruit beta-arrestins and to undergo internalization in the absence of functional heterotrimeric G proteins (G-zero). On the other hand, at G-zero, ligand stimulation of various GPCRs did not lead to any detectable beta-arrestin-mediated cellular responses. These novel findings strongly suggest that beta-arrestin-mediated signaling requires the presence of functional heterotrimeric G proteins. (Grundmann M, Merten N, Malfacini D, Inoue A, Preis P, Simon K, Rttiger N, Ziegler N, Benkel T, Schmitt NK, Ishida S, Mller M, Reher R, Kawakami K, Inoue A, Rick U, Khl T, Imhof D, Aoki J, Knig GM, Hoffmann C, Gomeza J, Wess J, Kostenis E. Lack of beta-arrestin signaling in the absence of active G proteins. Nat Comm 9, no. 341, doi:10.1038/s41467-017-02661-3, 2018)